The objective of this proposal is to gain an understanding of the control mechanisms involved in eukaryotic gene regulation, using adenovirus type 2 (Ad2) early region 2 as the model system. This is to be accomplished by a combined genetic and biochemical approach. To this end, I will (1) construct a cell line that contains and constitutively expresses the early region 2 gene, (2) generate large numbers of point mutations and deletions by site-specific mutagenesis, using the newly constructed cell line as the permissive host, (3) analyze the effects of the mutations on the synthesis and function of the early region 2 gene product, and (4) sequence mutant virus DNAs to obtain information/function correlations. The early region 2 gene product of Ad2, a 72,000 dalton singled-stranded DNA-binding protein, is involved in at least four functions: (1) the initiation and (2) elongation of DNA synthesis, (3) the regulation of transcription of early RNAs, and (4) the host-range of the virus. The approach described in this proposal will allow determination of the residues of the protein involved in each of these functions, and will identify sequences in the DNA and RNA that are important for transcription and processing of the region 2 mRNA.